Distribution of blaOXA-10, blaPER-1, and blaSHV genes in ESBL-producing Pseudomonas aeruginosa strains isolated from burn patients

Pseudomonas aeruginosa is resistant to a wide range of extended spectrum-lactamases (ESBLs) antibiotics because it produces several kinds of ESBLs. The goal of the current investigation was to identify the bacteria that produce extended spectrum -lactamases and the genes that encode three different ESBLs, such as blaOXA-10, blaPER-1 and blaSHV genes in Pseudomonas aeruginosa isolated from burn patients. In this investigation, 71 Pseudomonas aeruginosa isolates were isolated from burn wounds in Burn and Plastic Surgery Hospital, Duhok City between July 2021 to June 2022. For the purpose of finding the blaOXA-10, blaPER-1, and blaSHV ESBL expressing genes, Polymerase Chain Reaction (PCR) was used. Among 71 Pseudomonas aeruginosa isolates, 26.36% (29/71) were isolated from males and 38.18% (42/71) from females, and 76.06% (54/71) of the isolates were multidrug resistant. They exhibited higher resistance against Piperacillin with resistance rates of 98.59%. Among the ESBL-producing isolates tested, blaOXA-10 was found in 59.26% (32), blaPER-1 was found in 44.44% (24), and blaSHV was found in 11.11% (6). All isolates must undergo antimicrobial susceptibility testing because only a few numbers of the available antibiotics are effective for the treatment of this bacterium. This will prevent the development of resistance in burn units and aids in the management of the treatment plan.

the cell or be transferred from cell to cell horizontally by conjugation, transformation or, in the case of bacteriophages, by transduction 12 .
Pseudomonas aeruginosa possesses a number of ESBLs, including those of the Ambler class A, which contain a variety of enzymes, such as Pseudomonas extended resistance bla (PER-1).In 1993, a Turkish patient being treated at a French hospital provided the first proof of PER-1, which only P. aeruginosa produces 13 .Displaying resistance to Cephalosporins and inhibition to Clavulanate, this enzyme hydrolyzed most Penicillins well and Cephalosporins including Cefalotin, Cefoperazone, Cefuroxime, Ceftriaxone, and Ceftazidime.PER-1 did not hydrolyse Oxacillin, Cephamycins or Imipenem.PER enzymes are most commonly found in isolates from Turkey and Mediterranean countries 14 .
Pseudomonas aeruginosa is the documented source of oxacillinase (OXA type), a class D -lactamase that hydrolyzes Oxacillin, but it has also been found in numerous other gram-negative bacteria, including Enterobacteriaceae.In general, OXA-type enzymes are a diverse category that exhibits variation in amino acid sequences and substrate profiles.However, it has been shown that a number of OXA-type variations hydrolyze cephalosporins, cephems, and/or monobactams.According to a recent review, there are 27 oxacillinase enzymes described as extended-spectrum.These enzymes' substrates include third-and/or fourth-generation Cephalosporins in addition to Penicillins and early Cephalosporins.Most extended-spectrum oxacillinases derive from OXA-10 and OXA-2.The OXA-10 derivatives include OXA-11, OXA-13, OXA-14, OXA-16, OXA-17, OXA-19 and OXA-28 15 .In general, -lactamase inhibitors have no effect on OXA-type enzymes 16 .The occurrence of SHV-type ESBLs has been recorded in a number of European nations, including Austria, France, Italy, and Greece, as well as the United States and Australia.Perhaps more so than any other form of ESBL, they will be found in clinical isolates 17 .
The goal of the current investigation was to identify the patterns of antibiotic susceptibility against various antibiotics because P. aeruginosa is becoming increasingly resistant to multiple ESBLs.In addition, genes encoding bla OXA-10 , bla PER-1 , and bla SHV were analyzed in clinical isolates of P. aeruginosa from infected hospitalized burn patients to assess their prevalence.

Results
In this current study, a total of 110 burn patient samples of both genders and different ages were collected from July 2021 to May 2022.From 110 samples, P. aeruginosa was the predominant pathogenic bacteria, and 71 (64.55%) of them were confirmed phenotypically and molecularly by the species-specific gene (16S rDNA).Phenotypically, P. aeruginosa was identified on MacConkey, Cetrimide, Nutrient, and Blood agars as well as Gram stain and biochemical tests (Oxidase, Citrate, Catalase, TSI).In addition, all isolated P. aeruginosa were genotypically confirmed by the species-specific gene (16S rDNA).
Among 71 P. aeruginosa, 26.36% (29/71) were isolated from males and 38.18% (42/71) from females.There was a statistically non-significant (P > 0.05) difference between both genders.Most isolates were collected from ages between 21 and 30 years (13.64%).Statistical analysis revealed that there were significant (P < 0.04) differences across age groups.The demographic information for 71 positive patients from whom P. aeruginosa was isolated are displayed in Table 1.The most frequent types of burn wounds were those caused by flame, accounting for 38.18% (42/110) of the patients; electric burns affected 2.73% (3/110) of the patients.Statistical analysis showed highly significant (P < 0.01) differences in the causes of burn.Patients with second-degree burns had a higher risk of contracting infection (45.45%) than third-and mix-degree burns (6.36% and 12.73%) respectively.

Discussion
Recently, ESBL-producing P. aeruginosa has become a significant source of infections in healthcare settings, particularly in immunocompromised individuals and burn victims 18 .Because the surface of moist wounds provides a habitat that is suited for their existence, it thrives in the clinical setting, proving that this environment is perfect for their colonization.Because of the high levels of resistance to the most often given antibiotics in hospital settings, the treatment of infections brought on by these multidrug-resistant organisms is becoming more difficult 19 .
Our study revealed the prevalence of P. aeruginosa isolates among burn patients at the Duhok Burn Hospital, at a rate of 64.55% (71/110), a similar finding was made in an Algerian study, where the rate was recorded at  62% 20 .On the other hand, another study conducted in Iraq recorded a high prevalence of P. aeruginosa isolates at 97.6% 21 .however, results from other studies from other nations showed lower prevalence rates, including Morocco (15.1%) 22 , and Egypt (19.8%) 23 .This difference might be attributed to antibiotics abuse, different hospital strategies for the management of infections, hygiene, and geographic climatic.
The current study showed the highest rate of 38.18% from flame burn followed by scald burn (15.45%).Regarding gender and age, females showed the highest rate of burn wounds than males (38.18% vs 26.36%), with the ages of 21-30 showing the highest rate (13.64%).These results are somewhat similar to the study conducted in Basra/Iraq 24 they showed that females had a higher proportion of burns than males (57.5% vs. 19.16%),with flame burns accounting for 76.6%, followed by scald burns (19.1%).Additionally, studies from Iraq and Iran have reported somewhat lower rates; 57% in the Iraqi city of Suleimani 25 , 54.84%, and 56% in Iran 1,26 respectively.Due to the fact that most flame injuries occur at home and that most women in our society perform daily household tasks like heating and cooking in areas of the kitchen where there is a greater danger of flame burns, females were significantly more likely than males to sustain flame burns.
While a study conducted in Suleimani/Iraq showed the highest infection in males (56.2%) with scald (Hot water) being the highest (72.5%) group involved, followed by flame (22.8%) 27 .According to this research's findings, the highest rate 45.45% were second-degree burn, followed by 12.73% of mixed burn infections and only 6.36% had third-degree burns.Similarly, Al-Aali et al. 3 revealed that 72.1% of burn patients had second-degree burns.According to the present study observations, patients who had TBSA burns were 20-40% more likely to contract an infection (48.18%), this finding is consistent with the research conducted by Rashid et al. 27 in Suleimani/Iraq found infection rates were higher in patients with TBSA burns of 20-40% compared to those with burns of 40%.The higher the total body surface area damaged by the thermal assault the higher the potential for the bacteria to colonize and proliferate increasing the wound thickness and depth making way to the bloodstream involvement 28 .
Regardless to the etiology of the burns, female patients over the age of ten years were the most prevalent patient demographics linked to a higher prevalence of P. aeruginosa infection.In the present investigation, 76.06% of the patients had MDR P. aeruginosa infection which is higher than that reported by other studies in Iran (16.5-41%),Iraq (12.4%) [29][30][31] , Brazil (71.4%) and Egypt (70%) 32,33 .On the other hand, another study in Iran found a much higher rate (89.24%) of patients infected with MDR P. aeruginosa 26 .Worldwide increasing of MDR P. aeruginosa could be attributed to improper use of antibiotics in hospitals and communities in addition to the accumulation of a variety of resistance mechanisms 34 .www.nature.com/scientificreports/ In the current investigation, 100% of the isolates demonstrated sensitivity to Colistin.In contrast, a substantially lower percentage of Colistin-sensitive individuals (53.1%) were reported by Jalil et al. 24 in their investigation in Iraq.Another study in Iraq also reported resistance to colistin at a rate of 7.4% 21 .However, susceptibility to Colistin remains vastly high against P. aeruginosa approaching 100% in most countries in the area of the Middle East and North Africa 35 .
In this study, 60.56% of P. aeruginosa isolates were resistant to Imipenem, which is higher than that reported by a study conducted in Iran at a rate of 41.3% 17 and Iraq at a rate of 47% 36 , and lower than that reported by studies conducted in Iran at a rate (83.9%) 26 , in Iraq 68.40% 21 .Multifactorial agents, such as increased carbapenemase production, oprD mutation, AmpC, and efflux pumps overexpression, a shift in drug target sites, and other mechanisms, are contributing to P. aeruginosa increasing resistance to carbapenems 37 .
The current study reported the highest resistance of P. aeruginosa to Piperacillin (98.59%).This finding is higher than previous studies conducted in Iran 74.8% 38 and in South Africa, 94% 39 .These findings raise a major concern that requires Health Authorities to urgently work on finding rapid and accurate diagnostic procedures and regulating the dispensing of antibiotics, in addition to tightening microbiological control systems in hospitals.
ESBLs are one of the most common sources of ß-lactam antibiotic resistance in Gram-negative bacteria.These enzymes are plasmid-encoded ß-lactamases that have been discovered in Klebsiella pneumoniae and Escherichia coli, as well as clinical isolates of Enterobacteriaceae and Pseudomonas 19 .In the current study, the genes linked to bla OXA-10 , bla PER-1 , and bla SHV were found in 59.26% (32), 44.44% (24), and 11.11% (6) of the ESBL-producing P. aeruginosa isolates.The level of resistance was even higher in isolates carrying the OXA-10 gene.The results were consistent with a study conducted in Iran by Farshadzadeh 40 , which used the same primers for the OXA-10 and PER-1 genes and produced a band of the same molecular weight.Another study conducted in Shiraz, Iran reported a rate of 76.2% bla OXA-10, and 40% of resistant isolates contain bla PER-1 -related genes 13 .The prevalence of the bla SHV gene in the current study is consistent with the rate of 10.7% reported by Peymani et al. 17 in Iran.
Different environments may have a different distribution of ESBL-resistant bacteria, as evidenced by the varying prevalence of ESBL-encoding genes.In addition, the prevalence of bla PER-1 in Ahvaz, Iran, was 62.75%, which was higher than the prevalence of bla PER-1 in Italy (34.61%) 41 , Hungary (1.3%), and Belgium (2%), respectively 42 , but less than the incidence of bla PER-1 in Turkey (86.75%) 43 .The results of our study showed a high prevalence of the ESBL-producing P. aeruginosa isolates in burn patients, which is an alarming sign and should be taken into consideration because increasing of the antimicrobial-resistant bacteria isolated from burn patients is an important issue.

Statistical analysis
Statistical analysis was performed using GraphPad Prism version 9.3.1 (471).The chi-square test and Odd (ratio 95% CI) test were performed to determine statistical significance at P-value of less than 0.05.

Conclusion
In conclusion, the current study's high proportion of ESBL-producing P. aeruginosa highlights a serious health issue that requires further attention.All isolates must undergo an antimicrobial susceptibility test prior to therapy because there are only a small number of medications that are effective against this bacterium.The execution of this test reduces the indiscriminate use of antibiotics and resistance development in burn units and aids in the management of treatment plan.

Table 2 .
Distribution of burnt TBSA and depth (degree) in relation to infection.

Table 4 .
Representation of the antibiotic resistance of ESBL-producing P. aeruginosa.

Table 5 .
Frequency of ESBLs producing strains and susceptibility patterns to antimicrobial agents in relation to bla OXA-10 , bla PER-1 and bla SHV.

Table 6 .
Molecular weights of the genes and sequences of the primer used.

Table 7 .
PCR program that applies in the thermo-cycles.